Assessing the risk of a major adverse cardiac event in patients with chest pain

ABSTRACT

Methods for characterizing the near term risk of experiencing a major adverse cardiac event in a patient presenting with chest pain are provided. In one embodiment the method comprises determining the level of myeloperoxidase (MPO) activity in a bodily sample obtained from the patient. In another embodiment, the method comprises determining the level of MPO mass in a bodily sample obtained from the patient. In another embodiment, the method comprises determining the level of one or more select MPO-generated oxidation products in a bodily sample obtained from the patient. The select MPO-generated oxidation products are dityrosine, nitrotyrosine, chlorotyrosine, methionine sulphoxide or an MPO-generated lipid peroxidation product. Levels of MPO activity, MPO mass, or the select MPO-generated oxidation product in bodily samples from the test subject are then compared to a control value that is derived from measurements of MPO activity, MPO mass, or the select MPO-generated oxidation product in comparable bodily samples obtained from control population. Such comparison can also be used to determine treatment of the patient immediately at presentation in the emergency room.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/513,490 filed on Oct. 22, 2003, which is incorporated hereinby reference in its entirety.

STATEMENT ON FEDERALLY FUNDED RESEARCH

This invention is supported, at least in part, by, by Grant No. RO1HL62526-01 from the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Clinical, electrocardiographic criteria, and conventional laboratorytesting have been used to evaluate patients who experience chest pain.However, these procedures do not adequately predict risk of experiencingan adverse cardiac event for patients presenting with acute coronarysyndromes. The use of C-reactive protein and other biomarkers have beenadvocated to more accurately gauge risk, but additional prognostic toolspredicting coronary artery vulnerability for near-term subsequent majorevents in patients who present with suspected acute coronary syndromesare needed.

Creatinine kinase isoenzymes and/or cardiac troponins, are used asdiagnostic biomarkers of myocardial necrosis in patients who presentwith chest pain.^(4,5) However, many patients who present with chestpain have normal levels of creatinine kinase isoenzymes or troponins,but subsequently experience a myocardial infarction, requirerevascularization, or die in the ensuing 6 months. Accordingly,additional biochemical markers for determining whether a patient whopresents with chest pain is at risk of experiencing a major adversecardiac event are required. Biochemical markers that can be used todetermine whether a patient who presents with chest pain is at risk ofrequiring medical intervention near term, e.g. within the next one tosix months are especially desirable.

SUMMARY OF THE INVENTION

The present invention provides methods of determining whether a patientwho presents with chest pain is at risk near term of experiencing amajor adverse cardiac event, including, but not limited to, myocardialinfarction, reinfarction, or death. The present invention also providesmethods of determining whether a patient who presents with chest painsis at risk near term of requiring medical intervention, including butnot limited to, urgent cardiac catherization, stress testing, coronarypulmonary bypass surgery, revascularization, etc.

In one embodiment, the method comprises determining the level of MPOactivity in blood or a derivative thereof, including but not limited to,leukocytes, neutrophils, monocytes, serum, or plasma of a patient whopresents with chest pain. The level of MPO activity in the patient'sblood is then compared to a control value that is derived frommeasurements of MPO activity in comparable bodily samples obtained froma control population, such as the general population, or a selectpopulation of human subjects, such as patients who have presented withchest pain but who have not experienced a major adverse cardiac eventwithin 6 months after presenting with chest pain. Such comparison isthen used to characterize the patient's risk of experiencing a majoradverse cardiac event, such as a myocardial infarction, reinfarction,the need for revascularization, or death. The present invention isparticularly useful for characterizing the patient's risk ofexperiencing such a major adverse cardiac event near term, e.g. withinthe following 1 to 6 months. For example, patients whose blood levels ofMPO activity are higher than the control value are at greater risk ofexperiencing a major cardiac event than patients whose blood MPOactivity levels are at or close to the control value. Moreover, theextent of the difference between the patient's MPO activity levels andcontrol value is also useful for characterizing the extent of the riskand for determining which patients would most greatly benefit fromcertain medical interventions, such as revascularization.

In another embodiment, the method comprises determining the level of MPOmass in a bodily sample obtained from the patient who is presenting withchest pain. The bodily sample is blood or a derivative thereof,including but not limited to, leukocytes, neutrophils, monocytes, serum,or plasma. Levels of MPO mass in bodily samples from the patient arethen compared to a control value that is derived from measurements ofMPO mass in comparable bodily samples obtained from a control populationas, such as the general population, or a select population of humansubjects, such as patients who have presented with chest pain but whohave not experienced a major cardiac event within 6 months afterpresenting with chest pain.

In another embodiment, the method comprises determining the level of oneor more select MPO-generated oxidation products in a bodily sampleobtained from the test subject. The select MPO-generated oxidationproducts are dityrosine, nitrotyrosine, chlorotyrosine, methioninesulphoxide, and MPO-generated lipid peroxidation products. MPO lipidperoxidation products include, but are not limited to,hydroxy-eicosatetraenoic acids (HETEs); hydroxy-octadecadienoic acids(HODEs); F₂Isoprostanes; the glutaric and nonanedioic monoesters of2-lyso phosphatidylcholoine (G-PC and ND-PC, respectively); the9-hydroxy-10-dodecenedioic acid and 5-hydroxy-8-oxo-6-octenedioic acidesters of 2-lysoPC(HDdiA-PC and HOdiA-PC, respectively); the9-hydroxy-12-oxo-10-dodecenoic acid and 5-hydroxy-8-oxo-6-octenoic acidesters of 2-lysoPC(HODA-PC and HOOA-PC, respectively); the9-keto-12-oxo-10-dodecenoic acid and 5-keto-8-oxo-6-octenoic acid estersof 2-lysoPC (KODA-PC and KOOA-PC, respectively); the9-keto-10-dodecendioic acid and 5-keto-6-octendioic acid esters of2-lysoPC (KDdiA-PC and KOdiA-PC, respectively); the 5-oxovaleric acidand 9-oxononanoic acid esters of 2-lysoPC (OV-PC and ON-PC,respectively); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide);5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide);5-cholesten-3β,7β-diol (7-OH-cholesterol); 5-cholesten-3β, 25-diol(25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOHcholesterol); and cholestan-3β, 5α, 6β-triol (triol). The bodily sampleis blood, urine or a blood derivative, including but not limited to,leukocytes, neutrophils, monocytes, serum, or plasma. Levels of theselected MPO-generated oxidation products in bodily samples from thepatient who presents with chest pain are then compared to a controlvalue or control range of values that is derived from measurements ofthe selected MPO-generated oxidation products in comparable bodilysamples obtained from a control population, such as the generalpopulation, or a select population of human subjects, such as patientswho have presented with chest pain but who have not experienced a majorcardiac event within 6 months after presenting with chest pain.

Also provided are kits which are employed in the present methods.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Risks for Cardiovascular Outcomes in Troponin Negative PatientsBased Upon Baseline Myeloperoxidase Levels. Myeloperoxidase odds ratiosand 95 percent confidence intervals for revascularization and majoradverse cardiac outcomes in the ensuing 30 days and 6 months followingpresentation are shown. (circle) Unadjusted odds ratio associated witheach myeloperoxidase quartile; (square) adjusted odds ratio (for age,gender, C-reactive protein level and history of hyperlipidemia, historyof revascularization, history of myocardial infarction, and ECG changesconsistent with diagnosis of acute coronary syndrome) are shown.

FIG. 2. Receiver Operating Characteristic Curve Analyses of BiochemicalMarkers for Diagnosis of Acute Coronary Syndrome and 30 day CumulativeMajor Adverse Cardiac Events. Shown are receiver operatingcharacteristic curves for Troponin-T (maximum value), creatine kinase-MB(CK-MB, maximum value), C-reactive protein (CRP) and myeloperoxidase(MPO) for all patients (left panel) and for troponin negative patients(right panel).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with occasional reference tothe specific embodiments of the invention. This invention may, however,be embodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to that this invention belongs. The terminology used in thedescription of the invention herein is for describing particularembodiments only and is not intended to be limiting of the invention. Asused in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless otherwise indicated, the numerical properties setforth in the following specification and claims are approximations thatmay vary depending on the desired properties sought to be obtained inembodiments of the present invention. Notwithstanding that the numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values, however,inherently contain certain errors necessarily resulting from error foundin their respective measurements.

The disclosure of all patents, patent applications (and any patents thatissue thereon, as well as any corresponding published foreign patentapplications), GenBank and other accession numbers and associated data,and publications mentioned throughout this description are herebyincorporated by reference herein. It is expressly not admitted, however,that any of the documents incorporated by reference herein teach ordisclose the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless otherwise indicated, the numerical properties setforth in the following specification and claims are approximations thatmay vary depending on the desired properties sought to be obtained inembodiments of the present invention. Notwithstanding that the numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values, however,inherently contain certain errors necessarily resulting from error foundin their respective measurements.

Provided herein are methods for assessing the near term risk ofexperiencing a major cardiac event in a patient with chest pain.Provided herein are methods for determining whether a patient presentingwith chest pain is at near term risk of requiring medical intervention,such as revascularization. In one embodiment, the method comprisesobtaining the level of MPO activity in a blood sample obtained from thepatient presenting with chest pain. In another embodiment, the methodcomprises obtaining the level of MPO mass in a bodily sample from thepatient presenting with chest pain. In another embodiment, the methodcomprises obtaining the level of one or more select MPO-generatedoxidation products in a bodily sample from the patient presenting withchest pain. Such MPO-generated oxidation products are selected from thegroup consisting of dityrosine, nitrotyrosine, methionine sulphoxide,chlorotyrosine and a lipid peroxidation product. In yet anotherembodiment, the method comprises obtaining the level of MPO activity, orMPO mass, or both, and the level of one or more select MPO-generatedoxidation products in a bodily sample obtained from the patient.

The level of the selected risk predictor, i.e., MPO activity, MPO mass,MPO-generated oxidation product, or combinations thereof, in thepatient's bodily sample is then compared to a control value or range ofcontrol values that is based on the level of the same risk predictor ina comparable bodily sample of individuals in a control population. Suchcomparison provides information which characterizes the patient's risknear term of experiencing a major adverse cardiac event such asmyocardial infarction, or death. The difference between the level ofrisk predictor in the bodily sample of the patient and the level of therisk predictor in a bodily sample from individuals in the controlpopulation also provides information about the extent of the risk. Thus,the level of MPO or MPO-generated oxidation product in the blood of apatient presenting with chest pain can be used to identify patients inneed of more aggressive carepath and global risk reduction therapies,such as more urgent cardiac catherization or stress testing before beingdischarged to follow up with the patient's primary care physician.

The present invention also relates to kits that comprise assays for MPOactivity or mass, or the select MPO-generated oxidation product. Suchassays have appropriate sensitivity with respect to control valuesselected on the basis of the present predictive tests. The present kitsdiffer from those presently commercially available for MPO by including,for example, different cut-offs, different sensitivities at particularcut-offs, as well as instructions or other printed material forcharacterizing the patient's risk of experiencing a major adversecardiac event near term based upon the outcome of the assay.

Preparation of Bodily Sample

Whole blood is obtained from the patient using standard clinicalprocedures. Plasma is obtained from whole blood samples bycentrifugation of anti-coagulated blood. Such process provides a buffycoat of white cell components and a supernatant of the plasma.

Serum is collected by centrifugation of whole blood samples that havebeen collected in tubes that are free of anti-coagulant. The blood ispermitted to clot prior to centrifugation. The yellowish-reddish fluidthat is obtained by centrifugation is the serum.

Leukocytes can be isolated from whole blood samples by any of varioustechniques including buoyant density centrifugation as described in theexamples below.

Methods of Determining MPO Activity

MPO (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) is atetrameric, heavily glycosylated, basic (PI. 10) heme protein ofapproximately 150 kDa. It is comprised of two identical disulfide-linkedprotomers, each of which possesses a protoporphyrin-containing 59-64 kDaheavy subunit and a 14 kDa light subunit (Nauseef, W. M, et al., Blood67:1504-1507; 1986.)

Myeloperoxidase activity may be determined by any of a variety ofstandard methods known in the art. One such method is acolorimetric-based assay where a chromophore that serves as a substratefor the peroxidase generates a product with a characteristic wavelengthwhich may be followed by any of various spectroscopic methods includingUV-visible or fluorescence detection. Additional details of colorimetricbased assays can be found in Kettle, A. J. and Winterboum, C. C. (1994)Methods in Enzymology. 233: 502-512; and Klebanoff, S. J., Waltersdorph,A. N. and Rosen, H. (1984) Methods in Enzymology. 105: 399-403, both ofwhich are incorporated herein by reference. An article by Gerber,Claudia, E. et al, entitled “Phagocytic Activity and Oxidative Burst ofGranulocytes in Persons with Myeloperoxidase Deficiency” published in1996 in Eur. J. Clin. Chem. Clin Biochem 34:901-908, describes a methodfor isolation for polymorphonuclear leukocytes (i.e. neutrophils) andmeasurement of myeloperoxidase activity with a colorometric assay, whichinvolves oxidation of the chromgen 4-chloro-1-naphthol.

Peroxidase activity may be determined by in situ peroxidase staining inMPO containing cells with flow cytometry-based methods. Such methodsallow for high through-put screening for peroxidase activitydeterminations in leukocytes and subpopulations of leukocytes. Anexample is the cytochemical peroxidase staining used for generatingwhite blood cell count and differentials with hematology analyzers basedupon peroxidase staining methods. For example, the Advia 120 hematologysystem by Bayer analyzes whole blood by flow cytometry and performsperoxidase staining of white blood cells to obtain a total white bloodcell count (CBC) and to differentiate amongst the various white bloodcell groups.

With these methods, whole blood enters the instrument and red bloodcells are lysed in a lysis chamber. The remaining white blood cells arethen fixed and stained in situ for peroxidase activity. The stainedcells are channeled into the flow cytometer for characterization basedupon the intensity of peroxidase staining and the overall size of thecell, which is reflected in the amount of light scatter of a given cell.These two parameters are plotted on the x and y axis, respectively, byconventional flow cytometry software, and clusters of individual cellpopulations are readily discernible. These include, but are not limited,to neutrophils, monocytes and eosinophils, the three major leukocytepopulations containing visible peroxidase staining.

During the course of these analyses, leukocytes such as monocytes,neutrophils, eosinophils and lymphocytes are identified by the intensityof peroxidase staining and their overall size. Information about theoverall peroxidase activity staining within specific cell populations isthus inherent in the position of individual cell clusters (e.g.neutrophil, monocyte, eosinophil clusters) and peroxidase levels withinspecific cell populations may be determined. Peroxidaseactivity/staining in this detection method is compared to a peroxidasestain reference or calibrant. Individuals with higher levels ofperoxidase activity per leukocyte are identified by having a cellpopulation whose location on the cytogram indicates higher levels ofperoxidase (i.e., average peroxidase activity per leukocyte) or bydemonstrating a sub-population of cells within a cell cluster (e.g.neutrophil, monocyte, eosinophil clusters) which contain higher levelsof peroxidase activity either on average or in a higher subgroup, suchas the higher tertile or quartile.

Methods of Determining MPO Mass

The mass of myeloperoxidase in a given sample is readily determined byan immunological method, e.g. ELISA. Commercial kits for MPOquantification by ELISA are available.

MPO mass in a sample can also be determined indirectly by in situperoxidase staining of the bodily sample. Methods which analyzeleukocyte peroxidase staining can be performed on whole blood, such asthose with hematology analyzers which function based upon in situperoxidase staining. Previous studies by other investigators havedemonstrated that the overall intensity of staining is proportional toperoxidase mass (e.g. Claudia E. Gerber, Selim Kuci, Matthias Zipfel,Ditrich Niethammer and Gernot Bruchfelt, “Phagocytic activity andphagocytic activity and oxidative burst of granulocytes in persons withmyeloperoxidase deficiency” European Journal of Clinical Chemistry andClinic Biochemistry (1996) 34: 901-908).

Flow cytometry through a hematology analyzer is a high through-puttechnique for quantifying the parameters used in determining MPOactivity or mass levels or numbers of cells containing elevated levelsof MPO activity or mass. The advantage of using such a technique is itsease of use and speed. The Advia 120 can perform 120 complete cell bloodcount and differentials in one hour and utilizes only a few microlitersof blood at a time. All the data necessary for determination of theperoxidase activity is held within the flow cytometry cell clusters usedto ultimately calculate the total white blood cell count anddifferential. With minor adjustments to software of this apparatus, thereadout can be modified to include multiple different indices of overallperoxidase activity. For example, patients presenting with chest painwhose neutrophil clusters contain an overall increase in the averageperoxidase activity (i.e. increased mean peroxidase index) will be atincreased risk for experiencing a major adverse cardiac event. Inaddition to simply determining the mean peroxidase activity for a givencell type, individuals at increased risk of experiencing a major adversecardiac event can be identified by examining the overall distribution ofperoxidase activity within a given cell cluster (mean+mode, etc). It isexpected that by looking at the population of peroxidase activity perleukocyte, patients who possess leukocytes with a higher proportion ofcells containing a high peroxidase activity in a subset of cells (forexample, the upper quartile, or the upper tertile) may be atparticularly high risk.

Levels of MPO Activity and MPO Mass

The level of MPO activity or MPO mass in the body fluid can bedetermined by measuring the MPO activity or MPO mass in the body fluidand normalizing this value to obtain the MPO activity or mass per ml ofblood, per ml of serum, per ml of plasma, per leukocyte (e.g. neutrophilor monocyte), per weight, e.g. mg of total blood protein, per weight ofleukocyte protein (e.g. per weight of neutrophil or monocyte protein).Alternatively, the level of MPO activity or MPO mass in the body fluidcan be a representative value which is based on MPO activity in the testsubjects blood or blood derivatives. For example the level of MPOactivity can be the percentage or the actual number of the testsubject's neutrophils or monocytes that contain elevated levels of MPOactivity or MPO mass. Examples of other representative values include,but are not limited to, arbitrary units for a parameter that can beobtained from a flow cytometry based cytogram, such as the position ofthe neutrophil cluster on the X and Y axes, or the angle of the majoraxis of the neutrophil cluster relative to the X and Y axes.

Methods of Determining Levels of Select Myeloperoxidase-GeneratedOxidation Products Dityrosine and Nitrotyrosine

Dityrosine and nitrotyrosine levels in the bodily sample can bedetermined using monoclonal antibodies that are reactive with suchtyrosine species. For example, anti-nitrotyrosine antibodies may be madeand labeled using standard procedures and then employed in immunoassaysto detect the presence of free or peptide-bound nitrotyrosine in thesample. Suitable immunoassays include, by way of example,radioimmunoassays, both solid and liquid phase, fluorescence-linkedassays or enzyme-linked immunosorbent assays. Preferably, theimmunoassays are also used to quantify the amount of the tyrosinespecies that is present in the sample.

Monoclonal antibodies raised against the dityrosine and nitrotyrosinespecies are produced according to established procedures. Generally, thedityrosine or nitrotyrosine residue, which is known as a hapten, isfirst conjugated to a carrier protein and used to immunize a hostanimal. Preferably, the dityrosine and nitrotyrosine residue is insertedinto synthetic peptides with different surrounding sequence and thencoupled to carrier proteins. By rotating the sequence surrounding thedityrosine and nitrotyrosine species within the peptide coupled to thecarrier, antibodies to only the dityrosine and nitrotyrosine species,regardless of the surrounding sequence context, are generated. Similarstrategies have been successfully employed with a variety of other lowmolecular weight amino acid analogues.

Suitable host animals, include, but are not limited to, rabbits, mice,rats, goats, and guinea pigs. Various adjuvants may be used to increasethe immunological response in the host animal. The adjuvant useddepends, at least in part, on the host species. To increase thelikelihood that monoclonal antibodies specific to the dityrosine andnitrotyrosine are produced, the peptide containing the respectivedityrosine and nitrotyrosine species may be conjugated to a carrierprotein which is present in the animal immunized. For example, guineapig albumin is commonly used as a carrier for immunizations in guineapigs. Such animals produce heterogenous populations of antibodymolecules, which are referred to as polyclonal antibodies and which maybe derived from the sera of the immunized animals.

Monoclonal antibodies, which are homogenous populations of an antibodythat binds to a particular antigen, are obtained from continuous cellslines. Conventional techniques for producing monoclonal antibodies arethe hybridoma technique of Kohler and Millstein (Nature 356:495-497(1975)) and the human B-cell hybridoma technique of Kosbor et al(Immunology Today 4:72 (1983)). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, Iga, IgD and any classthereof. Procedures for preparing antibodies against modified aminoacids, such as for example, 3-nitrotyrosine are described in Ye, Y. Z.,M. Strong, Z. Q. Huang, and J. S. Beckman. 1996. Antibodies thatrecognize nitrotyrosine. Methods Enzymol. 269:201-209.

In general, techniques for direct measurement of protein bounddityrosine and nitrotyrosine species from bodily fluids involves removalof protein and lipids to provide a fluid extract containing free aminoacid residues. The tissues and bodily fluids are stored, preferably inbuffered, chelated and antioxidant-protected solutions, preferably at−80° C. as described above. The frozen tissue, and bodily fluids arethen thawed, homogenized and extracted, preferably with a single phasemixture of methanol:diethylether:water as described above to removelipids and salts. Heavy isotope labeled internal standards are added tothe pellet, which, preferably, is dried under vacuum, hydrolyzed, andthen the amino acid hydrolysate resuspended, preferably in awater:methanol mixture, passed over a mini solid-phase C18 extractioncolumn, derivatized and analyzed by stable isotope dilution gaschromatography-mass spectrometry as above. Values of free dityrosine andnitrotyrosine species in the bodily sample can be normalized to proteincontent, or an amino acid such as tyrosine as described above.

In a highly preferred procedure, protein is delipidated and desaltedusing two sequential extractions with a single phase mixture ofH₂O/methanol/H₂O-saturated diethyl ether (1:3:8 v/v/v). Oxidizedtyrosine standards (2 pmol each) and universal labeled tyrosine (2 nmol)are added to protein pellets. Proteins are hydrolyzed by incubating thedesalted protein pellet with degassed 6N HCl supplemented with 1% phenolfor 24 h under argon atmosphere. Amino acid hydrolysates are resuspendedin chelex treated water and applied to mini solid-phase C18 extractioncolumns (Supelclean LC-C18SPE minicolumn; 3 ml; Supelco, Inc.,Bellefone, Pa.) pre-equilibrated with 0.1% trifluoroacetic acid.Following sequential washes with 2 ml of 0.1% trifluoroacetic acid,oxidized tyrosines and tyrosine are eluted with 2 ml 30% methanol in0.1% trifluoroacetic acid, dried under vacuum and then analyzed by massspectrometry.

Tandem mass spectrometry is performed using electrospray ionization anddetection with an ion trap mass spectrometer (LCQ Deca, ThermoFinigann,San Jose, Calif.) interfaced with a Thermo SP4000 high performanceliquid chromatograph (HPLC). Samples are suspended in equilibrationsolvent (H₂O with 0.1% formic acid) and injected onto a Ultrasphere C18column (Phenominex, 5 μm, 2.0 mm×150 mm). L-Tyrosine and its oxidationproducts are eluted at a flow rate of 200 μl/min using a linear gradientgenerated against 0.1% formic acid in methanol, pH 2.5 as the secondmobile phase. Analytes are monitored in positive ion mode with full scanproduct ion MS/MS at unit resolution. Response is optimized with a sprayvoltage setting of 5 KV and a spray current of 80 μA. The heatedcapillary voltage is set at 10 V and the temperature to 350° C. Nitrogenis used both as sheath and auxiliary gas, at a flow rate of 70 and 30arbitrary units, respectively. The analyte abundance is evaluated bymeasuring the chromatographic peak areas of selected product ionsextracted from the full scan total ion chromatograms, according to thecorresponding ion trap product ion spectra. The ions monitored for eachanalyte are: 3-nitro[¹²C₆]tyrosine (mass-to-charge-ratio (m/z) 227, 181and 210), 3-nitro[¹³C₆]tyrosine (m/z 233, 187 and 216), 3-nitro[¹³C₉¹⁵N₁]tyrosine (m/z 237, 190 and 219), [¹²C₆]tyrosine (m/z 182, 136 and165), [¹³C₉ ¹⁵N₁]tyrosine (m/z 192, 145 and 174). Tyrosine andnitrotyrosine are base-line resolved under the HPLC conditions employed,permitting programming of the LCQ Deca for analysis over 0-7 min fordetection of tyrosine isotopomers, and from 7 min on for detection of3-nitrotyrosine isotopomers.

Free nitrotyorsine and dityrosine are similarly measured in samples, buttissue or bodily fluid is first passed through a low molecular weightcut off filter and the low molecular weight components analyzed byLC/ECS/MS/MS. Values of free and protein-bound dityrsoine andnitrotyrosine species in the bodily sample can be normalized to proteincontent, or an amicon acid such as the precursor tyrosine, as describedbelow.

Lipid Oxidation Products

Lipid oxidation products can be measured by HPLC with UV detection orHPLC with on line mass spectrometry. Other analytical methods includingGC/MS and immunocytochmeical methods may also be used. F₂Isoprostanesare measurable by various mass spectrometry techniques as known in theart.

Methods of extracting and quantifying the MPO-generated lipid oxidationproducts hydroxy-eicosatetraenoic acids (HETEs), hydroxy-octadecadienoicacids (HODEs), F2Isoprostanes; the 5-oxovaleric acid esters of 2-lysoPC(OV-PC); 5-cholesten-5α,6α-epoxy-3β-ol (cholesterol α-epoxide);5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide);5-cholesten-3β,7β-diol (7-OH-cholesterol); 5-cholesten-3β, 25-diol(25-OH cholesterol 5-cholesten-3β-ol-7β-hydroperoxide (7-OOHcholesterol); and cholestan-3β, 5α, 6β-triol (triol) are described inSchmitt, et al. (1999) Biochemistry, Vol. 38, 16904-16915, which isspecifically incorporated herein by reference. For determination of9-H(P)ODE, 9-H(P)ETE and F₂-isoprostanes, hydroperoxides in reactionmixtures are reduced to their corresponding hydroxides during extractionutilizing a modified Dole procedure in which the reducing agent,triphenylphosphine, is present (Savenkova, M. L., et al. (1994) J. Biol.Chem. 269, 20394-20400). These conditions also inhibit artifactualformation of isoprostanes and oxidized lipids. Lipids are dried underN₂, resuspended in isopropanol (2 ml) and then fatty acids released bybase hydrolysis with 1 N sodium hydroxide (2 ml) at room temperatureunder N₂ for 90 min. The samples are acidified (pH 3.0) with 2N HCl,known amounts of internal standards are added and free fatty acids areextracted twice with hexane (5 ml). The content of 9-H(P)ODEs,9-H(P)ETEs and F₂-isoprostanes are then determined by LC/MS/MS analysisas outlined below.

1-palmitoyl-2 oxovaleryl-sn-glycero-3-phosphatidyl choline (PoxvPC) isextracted by the same modified Dole procedure used for 9-H(P)ODE,9-H(P)ETE and F₂ isoprostane analyses as above, but omitting addition ofthe reductant, triphenylphosphine. Lipids are dried under N₂,resuspended in methanol and stored under argon at −70° C. untilsubsequent LC/MS analysis as outline below. Sterol oxidation productsare extracted by adding 4 M NaCl (150 μl) and acetonitrile (500 μl).Samples are vortexed, centrifuged, and the upper organic phase removed.Extracts are dried under N₂, resuspended in methanol, and stored underargon at −70° C. until analysis by HPLC with on-line mass spectrometricanalysis.

Mass spectrometric analyses are performed on a Quatro II triplequadruple mass spectrometer interfaced with an HP 1100 HPLC.F₂-isoprostanes are quantified by stable isotope dilution massspectrometry using on-line reverse phase HPLC tandem mass spectrometry(LC/MS/MS) with 8-epi-[²H₄]PGF_(2α) as standard as described by Mallat(Mallat, Z., et al. (1999) J. Clin. Invest. 103, 421-427). For 9-HODEand 9-HETE analyses, lipid extracts generated following base hydrolysisof reduced lipids (above) are dried under N₂ and reconstituted inmethanol. An aliquot of the mixture is then injected on an UltrasphereODS C18 column equilibrated and run under isocratic conditions employingmethanol:H₂O, (85:15, v/v) as solvent. Column eluent is split (930μl/min to UV detector and 70 μl/min to mass detector) and analyzed bythe mass spectrometer. LC/MS/MS analysis of 9-HODE, 9-HETE andF₂-isoprostanes in column effluents is performed using electrosprayionization mass spectrometry (ESI-MS) in the negative-ion mode withmultiple reaction monitoring (MRM) and monitoring the transitions m/z295→171 for 9-HODE; m/z 319→151 for 9-HETE; m/z 353→309 forF₂-isoprostanes; and m/z 357→313 for [²H₄]PGF_(2α).

Quantification of POxvPC is performed on lipid extracts utilizing HPLCwith on-line ESI-MS analysis in the positive ion mode and selected ionmonitoring at m/z 782 and m/z 594, respectively. An aliquot of lipidextract reconstituted in methanol (above) is mixed 0.1% formic acid inmethanol (mobile phase B) and loaded onto a Columbus C18 column (1×250mm, 5 μm, P. J. Cobert, St. Louis, Mo.) pre-equilibrated in 70% mobilephase B, 30% mobile phase A (0.1% formic acid in water) at a flow rateof 30 μl/min. Following a 3 min wash period at 70% mobile phase B, thecolumn is developed with a linear gradient to 100% mobile phase B,followed by isocratic elution with 100% mobile phase B. Externalcalibration curves constructed with authentic POxvPC are used forquantification. 7-OH cholesterol, 7-keto cholesterol, and 7-OOHcholesterol are resolved on an Ultrasphere ODS C18 column. The elutiongradient consisted of 91:9, acetonitrile:water+0.1% formate (v:v), andthe column washed between runs with acetonitrile+0.1% formate. Columneffluent is split (900 μl/min to UV detector and 100 μl/min to massdetector) and ionized by atmospheric pressure chemical ionization (APCI)in the positive-ion mode with selected ion monitoring. Identification of7-OH cholesterol is performed by demonstrating co-migration of ions withm/z 385.3 (M−H₂O)⁺ and m/z 367.3 (M−2H₂O)⁺ with the same retention timeas authentic standard. The integrated area of the ion current for thepeak monitored at m/z 367.3 is used for quantification. Identificationof 7-OOH cholesterol ois performed by demonstrating co-migration of ionswith m/z 401.3 (M−H₂O)⁺, m/z 383.3 (M−2H₂O)⁺ and m/z 367.3 (M−H₂O₂)⁺with the same retention time as authentic standard. The integrated areaof the ion current for the peak monitored at m/z 401.3 is used forquantification. Identification of 7-keto cholesterol is performed bydemonstrating co-migration of ions with m/z 401.3 (M+H)⁺ and m/z 383.3(M−H₂O)⁺ with the same retention time as authentic standard. Theintegrated area of the ion current for the peak monitored at m/z 401.3is used for quantification. External calibration curves constructed withauthentic 7-OH cholesterol, 7-OOH cholesterol and 7-keto cholesterol areused for quantification following preliminary APCI LC/MS experimentsdemonstrating identical results to those obtained by the method ofstandard additions. The retention times for 25-OH cholesterol, 5,6 α-and β-epoxides, and triol are determined by LC/MS analysis of authenticstandards.

Control Value

The level of MPO mass, MPO activity, or select MPO-generated oxidationproduct in the bodily sample obtained from the test subject is comparedto a control value or range of values. The control value or range ofvalues is based upon the levels of MPO activity, MPO mass, or selectMPO-generated oxidation product in comparable samples obtained from acontrol population, such as the general population or a selectpopulation of human subjects. For example, the select population may becomprised of apparently healthy subjects. “Apparently healthy”, as usedherein, means individuals who have not previously had any signs orsymptoms indicating the presence of atherosclerosis, such as anginapectoris, history of an acute adverse cardiovascular event such as amyocardial infarction or stroke, evidence of atherosclerosis bydiagnostic imaging methods including, but not limited to coronaryangiography. Apparently healthy individuals also do not otherwiseexhibit symptoms of disease. In other words, such individuals, ifexamined by a medical professional, would be characterized as healthyand free of symptoms of disease. In another embodiment, the controlpopulation may be a population of test subjects who have presented withchest pain but who have not experienced a major cardiac event within 6months of presenting with chest pain.

The control value is related to the value used to characterize the levelof MPO activity or MPO mass in the bodily sample obtained from the testsubject. Thus, if the level of MPO activity is an absolute value such asthe units of MPO activity per leukocyte or per ml of blood, the controlvalue is also based upon the units of MPO activity per leukocyte or perml of blood in individuals in the control population. Similarly, if thelevel of MPO activity or MPO mass is a representative value such as anarbitrary unit obtained from a cytogram, the control value or range ofvalues is also based on the representative value.

The control value can take a variety of forms. The control value can bea single cut-off value, such as a median or mean. The control value canbe established based upon comparative groups such as where the risk inone defined group is double the risk in another defined group. Thecontrol value can be a control range of values, for example, where thegeneral population is divided equally (or unequally) into groups, suchas a low risk group, a medium risk group and a high-risk group, or intoquadrants, the lowest quadrant being individuals with the lowest riskthe highest quadrant being individuals with the highest risk.

The control value can be derived by determining the level of MPOactivity or mass in the general population. Alternatively, the controlvalue can be derived by determining the level of MPO activity or mass ina select population, such as an apparently healthy nonsmoker population.For example, an apparently healthy, nonsmoker population may have adifferent normal range of MPO activity or MPO mass than will a smokingpopulation or a population whose member have had a prior cardiovasculardisorder. Accordingly, the control values selected may take into accountthe category in which an individual falls. Appropriate ranges andcategories can be selected with no more than routine experimentation bythose of ordinary skill in the art.

Control values of MPO activity or MPO mass, such as for example, meanlevels, median levels, or “cut-off” levels, are established by assayinga large sample of individuals in control population or populations andusing a statistical model such as the predictive value method forselecting a positivity criterion or receiver operator characteristiccurve that defines optimum specificity (highest true negative rate) andsensitivity (highest true positive rate) as described in Knapp, R. G.,and Miller, M. C. (1992). Clinical Epidemiology and Biostatistics.William and Wilkins, Harual Publishing Co. Malvern, Pa., which isspecifically incorporated herein by reference. A “cutoff” value can bedetermined for each risk predictor that is assayed. The standardizedmethod that was used in Example 1 below employs the guaiacol oxidationassay as described in Klebanoff, S. J., Waltersdorph, A. N. and Rosen,H. 1984. “Antimicrobial activity of myeloperoxidase”. Methods inEnzymology. 105: 399-403).

By selecting the appropriate control population, sample size, andstatistical model, one can obtain a control value that enables one toassess the risk of the patient experiencing a major adverse cardiacevent within a time period of days to months following the chest pain.Thus, the present method can be used to assess the patient's risk ofexperiencing a major cardiac event within one month, two months, threemonths, four months, five months, six months, etc. of experiencing thechest pain.

Comparison of MPO Activity and Mass Levels and Levels of SelectMPO-Generated Oxidation Products in the Bodily Sample from the PatientPresenting with Chest Pain to the Control Value.

The levels of each risk predictor, i.e., MPO activity, MPO mass andselect MPO-generated oxidation product, in a bodily sample from thepatient presenting with chest pain may be compared to a single controlvalue or to a range of control values. If the level of the present riskpredictor in the bodily sample of the patient presenting with chest isgreater than the control value or range of control values, the patientis at greater risk of experiencing a major adverse cardiac event thanother patients presenting with chest pain whose risk predictor levelsare comparable to or below the control value or at the lower end of thecontrol range of values. In contrast, if the level of the present riskpredictor in the bodily sample of the patient presenting with chest painis below the control value or at the lower end of the range of controlvalues, the patient presenting with chest pain is at a lower risk ofrequiring aggressive medical intervention that patients presenting withchest pain whose levels of the select risk predictor are comparable toor above the control value or at the upper end of the range of controlvalues. For example, a patient presenting with chest pain who has a muchhigher number of neutrophils or monocytes or both with elevated levelsof MPO activity or MPO mass as compared to the control value is at highrisk of experiencing a major adverse cardiac event, and a patientpresenting with chest pain who has a lower number of neutrophils ormonocytes or both with decreased or lower levels of MPO activity or MPOmass as compared to the control value is at low risk of experiencing amajor adverse cardiac event near term. The extent of the differencebetween the test subject's risk predictor levels and control value isalso useful for characterizing the extent of the risk and thereby,determining which individuals would most greatly benefit from certainaggressive therapies. In those cases, wherein the control value rangesare divided into a plurality of groups, such as the control value rangesfor individuals at high risk, average risk, and low risk, the comparisoninvolves determining into which group the patient's level of therelevant risk predictor falls.

The present tests are useful for determining if and when patientspresenting with chest pain should be further evaluated, such as beingsubjected to a stress tests or angiography, or should be scheduled formedical intervention before being discharged to the care of theirprimary care physician. For example, patients whose values of MPOactivity (U/mg PMN protein; or U/ml blood) are above a certain cutoffvalue, or are in the higher tertile or quartile of a “normal range,”could be identified as those in need of more extensive follow-ups oraggressive intervention such as coronary bypass surgery (CABG),percutaneous coronary intervention or positive catherization.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims which are appended hereto.

Methods

Study Design: Recruitment occurred as part of a study comparingtroponin-T vs. creatine kinase-MB isoform for the diagnosis ofmyocardial infarction.²¹ Patients presenting to the Emergency Departmentwith chest pain of suspected cardiac origin within 24 hours of onsetwere eligible.

Clinical Diagnosis: Myocardial infarction was defined by positivetroponin-T (≧0.1 ng/ml). Unstable angina was ascertained based on thepresence of angina at rest, sudden increase in episodes of previouslystable angina, ST segment depression, or T wave inversions, asdescribed.²¹ ECG data were verified independently by blinded ECG corefacility personnel. Diagnosis of acute coronary syndrome was adjudicatedbased on myocardial infarction or unstable angina, as defined byprotocol, and confirmed by chart review by a blinded investigator.

Outcome Definitions: Patients were assessed for major adverse cardiacoutcomes (myocardial infarction, reinfarction, need forrevascularization, and death). Medical record review and follow up phoneinterviews were performed for 30 day and 6 month outcomes. Need forrevascularization was defined as undergoing coronary artery bypasssurgery, percutaneous coronary intervention or positive catheterizationwith ≧2 lesions with >70% stenosis.

Healthy Volunteers: Sequential subjects responding to advertisements ina community newspaper were recruited. Subjects (age ≧21y) withouthistory or clinical evidence of coronary artery disease were eligible toparticipate. Population characteristics were: age (49±12.4y), malegender (54.8%), family history of coronary artery disease (44.3%),current smoking (36.5%) or history of diabetes (2.6%), hypertension(22.6%), or hyperlipidemia (69.6%). The Institutional Review Board atthe Cleveland Clinic Foundation approved the study protocol.

Biochemical Analyses: Troponin-T measures were performed on an ES300analyzer (Boehringer Mannheim, Indianapolis, Ind.). Baselinemyeloperoxidase levels were measured by ELISA (OXIS, Intl., Portland,Oreg.). Each plate included a standard curve with isolatedmyeloperoxidase (extinction coefficient of 178,000 M⁻¹cm⁻¹; ref. 22) andcontrols to correct for interplate variability. High sensitivityC-reactive protein measures were determined by nephelometry (DadeBehring, Deerfield, Ill.). Creatine kinase-MB mass was measured byimmunoassay (Abbott Laboratories, Abbott Park, Ill.).

Statistical Analysis: Patient characteristics are presented as eithermean (SD) or median (IQR) for continuous measures and as number(percentage) for categorical measures. Differences between outcomegroups and associations among categorical variables were assessed withWilcoxon Rank Sum test. Unadjusted trends were evaluated with theCochran-Armitage trend test. Correlations among continuous variableswere assessed with Spearman rank-correlation coefficient. Multivariatelogistic regression models (SAS version 8.0, SAS Institute, Inc., Cary,N.C.) were developed to calculate odds ratios and 95% confidenceintervals.

Results

Population Characteristics

The study population consisted of 604 patients presenting to theEmergency Department with complaint of chest pain (Table 1). The meantime from chest pain to presentation was 4.0 hours. Final diagnosesincluded myocardial infarction (24%), unstable angina (17%), suspectedcoronary syndrome (37%), and non-cardiac chest pain (22%). Outcomes (30day) included myocardial infarction (146 events), death (9),revascularization (189) and major adverse cardiac event (245).

Myeloperoxidase Levels in Healthy Volunteers and in Subjects Presentingto the Emergency Department with Chest Pain

Plasma levels of myeloperoxidase in subjects presenting with chest painranged from 0 to 4666 pM with a median of 198 pM and an interquartilerange of 119 pM to 394 pM. These levels were significantly higher thanthose observed in healthy volunteers (n=115; median 120 pM,interquartile range of 97 pM to 146 pM; P<0.001). Myeloperoxidase levelsin patients were correlated with peak troponin-T (r=0.21, P<0.001),C-reactive protein levels (r=0.10, P=0.01), and age (r=0.11, P=0.01),but not white blood cell count (P=0.11). Myeloperoxidase levels werehigher in males (median 213 pM versus 184 pM; P=0.05). Medianmyeloperoxidase levels did not differ by smoking status, history ofdiabetes, hypertension, myocardial infarction or coronary arterydisease, but were significantly higher in patients with a history ofeither hyperlipidemia (232 pM versus 181 pM; P<0.01) orrevascularization (234 pM versus 189 pM; P<0.01).

Baseline Myeloperoxidase Levels and Risks at Index Presentation

Myeloperoxidase levels were higher in patients who experienced amyocardial infarction within 16 hours of presentation (median, 320 pMversus 178 pM; P<0.001). Among patients with no biochemical evidence ofsignificant myocardial necrosis at entry (t=0 h), baselinemyeloperoxidase levels were significantly elevated in those thatsubsequently manifest a positive cardiac troponin-T level within theensuing 4 to 16 hours (median, 353 pM versus 309 pM; P<0.001).

The incidence of myocardial infarction increased with increasingquartile of myeloperoxidase (quartiles 1,2,3, and 4 rates of 13.9%,16.6%, 25.2% and 38.4%; P<0.001 for trend). Patients with initial (t=0h) negative troponin-T levels, but subsequent (t=4-16 h) positivecardiac enzymes, were more likely to be in the 3^(rd) and 4^(th)myeloperoxidase quartiles (quartiles 1, 2, 3, and 4 rates of 5.3%, 5.3%,8.0%, and 17.2%; P<0.001 for trend). Myeloperoxidase levels alsocorrelated with the frequency of adjudicated diagnosis of acute coronarysyndrome, increasing from 22.5% to 58.0% in quartiles 1 to 4 (P<0.001for trend).

Baseline Myeloperoxidase Levels: 30 Day and 6 Month Outcomes

Myeloperoxidase levels at presentation were higher in patients thatsubsequently experienced revascularization or major adverse cardiacevents (myocardial infarction, re-infarction, need for revascularizationand death) in the ensuing 30 day and 6 month period (P<0.001 for allcomparisons). Myeloperoxidase levels were also increased in thosepatients (n=34) that died within 6 months of presentation (270 pM vs.194 pM; P=0.05).

Myeloperoxidase levels were highest in patients presenting within 4.0 to9.6 hours following onset of symptoms (mean ±SE, 351±47 pM; P=0.041 andP=0.002 for comparisons versus subjects presenting <2.0 h or >9.6 hafter onset of symptoms, respectively). Myeloperoxidase levels remaineda robust predictor of outcomes across the distribution of times betweenonset of symptoms and time of blood draw. Moreover, plasmamyeloperoxidase levels in subjects presenting <2 hours of onset ofsymptoms (mean ±SE, 291±32 pM) were significantly higher than those inhealthy volunteers (P<0.001).

Myeloperoxidase as Independent Predictor of Short- and Near-Term Risks

Myeloperoxidase quartiles increasingly predicted risk for myocardialinfarction at presentation, both for the entire cohort and for thosepatients with initial (t=0 h) negative troponin-T values (Table 2;p<0.001). Myeloperoxidase also predicted risk for major adverse cardiacevents within 30 days and 6 months following index presentation (Table2; p<0.001). The unadjusted odds ratio (95% confidence interval) formajor adverse cardiac outcomes within 30 days and 6 months for thehighest quartile of myeloperoxidase plasma levels were 4.7(2.8-7.7) at30 days and 4.7(2.9-7.7) at 6 months, respectively (P<0.001 each).Similar odds ratios and 95% confidence intervals were observed forplasma myeloperoxidase levels as a predictor of revascularization atboth 30 days and 6 months (not shown). Stratification based on genderrevealed that while myeloperoxidase levels were lower in females(P=0.05), they predicted similar risks for both genders (myeloperoxidase4^(th) quartile odds ratio (95 percent confidence intervals) for 30 dmajor adverse cardiac events of 8.3(3.4 to 20.2) for females, and3.5(1.9 to 6.5) for males).

To ascertain whether plasma myeloperoxidase levels independently predictrisk of revascularization, myocardial infarction and major adversecoronary events, multivariate logistic regression models were used.Adjustments were made for variables associated with myeloperoxidaselevels or outcomes in univariate models (age, gender, C-reactiveprotein, history of hyperlipidemia, history of revascularization,history of prior myocardial infarction, or ECG changes consistent withacute coronary syndromes). Unadjusted versus adjusted odds ratios and95% confidence intervals for the entire cohort were virtually identical,confirming that elevated levels of myeloperoxidase served as anindependent predictor of increased risk for myocardial infarction, needfor revascularization and major adverse coronary outcomes within 30 dand 6 months following presentation (P<0.001 each).

Clinical Outcomes in Troponin Negative Patients

To test whether myeloperoxidase serves not only as a marker ofinflammation in response to myocardial necrosis, but also as a sensitivepredictor of the vulnerable plaque, we examined whether plasmamyeloperoxidase levels predicted risk among patients that present to theEmergency Department with chest pain, but in whom no evidence ofmyocardial necrosis is noted (i.e. troponin-T negative throughoutmonitoring period, t=0-16 h). Within this cohort (n=462),myeloperoxidase levels were significantly higher in patients withsubsequent major adverse cardiac events in the intervening 30 days and 6months compared to those who did not experience an event (e.g. median(interquartile range) for subjects with versus without 30 day majoradverse cardiac event of 268(152-444)pM versus 158(100-307) pM,respectively; P<0.001). Among subjects who were troponin-T negativethroughout the index presentation (t=0-16 h), the frequency of 30 dayand 6 month major adverse cardiac events increased with increasingmyeloperoxidase quartiles (P<0.001 for each trend).

For troponin negative patients, the risk for revascularization and majoradverse cardiac events at 30 days and 6 months following initialpresentation increased with increasing quartiles of myeloperoxidase(Table 2). FIG. 1 illustrates adjusted and unadjusted odds ratios and95% confidence intervals for myeloperoxidase quartiles as predictors ofsubsequent revascularization and major adverse cardiac events withinsubjects who remained troponin-T negative in the Emergency Department.Multivariate adjustment using factors associated with plasmamyeloperoxidase levels and outcomes in the cohort failed tosignificantly alter risks, confirming that plasma levels ofmyeloperoxidase serve as strong and independent predictors of 30 day and6 month risk for revascularization and major adverse coronary events(FIG. 1).

Comparison with Established Diagnostic and Prognostic Biomarkers

To evaluate whether plasma myeloperoxidase levels provide additive valueto C-reactive protein levels, parallel analyses were performed forC-reactive protein levels (Table 2). C-reactive protein predicted riskfor myocardial infarction at presentation, but was not predictive ofmajor adverse cardiac events in troponin negative patients.

Receiver operator characteristic curves for prediction of acute coronarysyndrome and major adverse cardiac events for the entire cohort, and thetroponin negative cohort, were examined (FIG. 2). In troponin negativesubjects, areas under the curve were highest for myeloperoxidasecompared to troponin (using values <0.01 ng/ml), peak creatine kinase-MBor C-reactive protein (P<0.001 for all comparisons for both outcomes).Using a cutpoint for myeloperoxidase (≧198 pM) derived from the receiveroperator characteristic curve for the entire cohort and 30d majoradverse cardiac events as the outcome, and established cutpoints fortroponin-T, creatine kinase-MB and C-reactive protein,²³ sensitivity,specificity, positive and negative predictive values were calculated fortroponin-T (58.0%, 100.0%, 100.0% and 77.7%), creatine kinase-MB (42.4%,94.7%, 84.6% and 70.7%), C-reactive protein (31.7%, 68.9%, 40.6% and60.0%) and myeloperoxidase (65.7%, 60.7%, 53.3% and 72.2%).

To evaluate the potential clinical utility of myeloperoxidase,comparisons of predicted positive versus negative test results fortroponin-T, creatine kinase MB, C-reactive protein and myeloperoxidasewere calculated (Table 3). Myeloperoxidase levels significantly enhancedidentification of patients at risk despite negative troponin-T levels atevaluation as compared to other markers (Table 3). The combination ofeither an elevated troponin-T or an elevated myeloperoxidasesignificantly improved the ability to identify subjects at risk for 30day major adverse cardiac events from 58.0% (troponin only) to 84.5%(troponin plus MPO; P<0.001). Among patients classified as having anegative troponin-T, 22.3% went on to experience a major adverse cardiacevent in the ensuing 30d period; however, using myeloperoxidase as anadditive screening test reduces that number to 14.8%, a 38% reduction(P<0.01 for comparison).

DISCUSSION

The results of the present study reveal that plasma myeloperoxidaselevels predict cardiovascular risks independent of C-reactive proteinand other markers of inflammation. An initial plasma myeloperoxidaselevel in patients presenting to the Emergency Department with chest painprovided information useful in determining risks for myocardialinfarction at presentation, need for revascularization, and majoradverse cardiac events over the next 6 months. Perhaps more importantly,even in patients who “ruled-out” for a myocardial infarction by serialtroponins, an elevated baseline plasma myeloperoxidase level waspredictive of subsequent major adverse cardiovascular outcomes.

Plasma myeloperoxidase levels correlated with troponin-T levels and werepredictive of acute myocardial infarction. However, whereas troponin-Ttakes 3 to 6 hours following myocardial injury to rise to measurablecirculating levels, myeloperoxidase levels were significantly elevatedat baseline in patients that presented (even within 2 h onset ofsymptoms) with negative cardiac enzymes but that later became positive.These findings suggest that myeloperoxidase levels may have utility inthe triage of patients in the Emergency Department, and that plasmamyeloperoxidase levels may be a marker of unstable angina precedingmyocardial necrosis, and therefore a predictor of vulnerable plaque.

Patients that present with chest pain but without evidence of myocardialnecrosis are a diagnostically challenging group for risk stratification,and one in which markers of vulnerable plaques are needed. Perhaps themost remarkable finding in the present study are that plasma levels ofmyeloperoxidase serve as an excellent predictor of risk even in patientswho remained troponin-T negative throughout the index presentation. Incontrast, C-reactive protein was not significantly predictive in thisgroup. C-reactive protein is reported to serve as a predictor ofshort-term risk for major adverse cardiac events in a subset of troponinnegative subjects with acute coronary syndromes—those with chest pain atrest.²⁴ However, a significant proportion of troponin negative patientsat presentation have more diagnostically challenging histories, andelevated levels of C-reactive protein are seen in less than 50% ofpatients with myocardial infarction not preceded by unstable angina.²⁵

The present studies suggest that myeloperoxidase serves as a marker ofthe vulnerable plaque. A unique feature of myeloperoxidase is theability to identify patients at near-term risk for major adverse cardiacevents independent of recent myocardial necrosis. The present studiesdemonstrate that addition of myeloperoxidase to initial riskstratification screens in patients presenting with chest pain canincrease the health care provider's ability to identify individuals atincreased risk who otherwise might not be identified without invasivediagnostic testing.

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TABLE 1 Patient Characteristics Myocardial Infarction at Evaluation NoYes Clinical Characteristics (n = 462) (n = 142) Age, yr; mean ± SD 61.4± 13.8 66.5 ± 12.8^(a) Male gender-no. (%) 254 (55.0) 100 (70.4)^(a) CADhistory-no. (%) 210 (48.3)  71 (51.8)  Revascularization-no. (%) 151(34.4)  51 (37.2)  Diabetes-no. (%) 108 (24.1)  52 (36.9)^(b)Hypertension-no. (%) 287 (64.1) 100 (70.9)  Hyperlipidemia-no. (%) 215(48.2)  83 (59.3)^(b) Current smoking-no. (%)  92 (21.9)  42 (30.9)^(b)Smoking history-no. (%) 259 (59.0)  92 (67.2)  ^(a)p ≦ 0.001 forcomparison with troponin negative subjects ^(b)p < 0.05 for comparisonwith troponin negative subjects

TABLE 2 Odds Ratio Associated with Major Adverse Cardiac Events PerMyeloperoxidase and C-Reactive Protein Quartiles MyeloperoxidaseQuartile C-Reactive Protein Quartile 1 2 3 4 1 2 3 4 MPO (pM) <119.4119.4-197.9 198.0-393.9 >394.0 <1.925 1.926-5.470 5.471-11.640 >11.640Index Presentation Myocardial Infarction 1.0 1.2 (0.7-2.3) 2.1(1.2-3.8)* 3.9 (2.2-6.8)**^(t) 1.0 1.9 (1.0-3.6)* 3.1 (1.7-5.8)** 3.0(1.6-5.4)*^(t) All patients TnT negative 1.0 1.0 (0.4-2.7) 1.5(0.6-3.9)3.7 (1.6-8.5)**^(t) 1.0 1.6 (0.7-3.6) 1.7 (0.8-3.9) 1.1 (0.5-2.7)initially Adjudicated ACS All patients 1.0 1.6 (1.0-2.7) 3.5 (2.1-5.8)*4.8 (2.9-7.8)* 1.0 1.7 (1.1-2.8)* 2.0 (1.2-3.2)* 1.5 (0.9-2.3) TnTnegative 1.0 2.0 (1.0-4.2) 4.6 (2.3-9.2)* 4.1 (2.0-8.5)* 1.0 1.5(0.8-2.6) 1.2 (0.6-2.1) 0.6 (0.3-1.3) patients MACE, 30 days Allpatients 1.0 1.7 (1.02-2.8)* 3.2 (2.0-5.4)** 4.7 (2.8-7.7)**^(t) 1.0 1.9(1.2-3.1)* 1.7 (1.0-2.7)* 1.6 (1.0-2.5) TnT negative 1.0 2.2 (1.1-4.6)*4.2 (2.1-8.4)** 4.1 (2.0-8.4)**^(t) 1.0 1.7 (1.0-3.0) 0.8 (0.4-1.6) 0.8(0.4-1.5) patients MACE, 6 months All patients 1.0 1.6 (1.0-2.7) 3.6(2.2-5.8)** 4.7 (2.9-7.7)**^(t) 1.0 1.8 (1.1-2.9)** 1.7 (1.1-2.7)* 1.8(1.1-2.8)*^(t) TnT negative 1.0 1.9 (1.0-3.8) 4.4 (2.3-8.4)** 3.9(2.0-7.7)**^(t) 1.0 1.6 (0.9-2.7) 0.9 (0.5-1.7) 1.0 (0.6-1.9) patients*p < 0.05 relative to first quartile; **p < 0.01 relative to firstquartile; ^(t)p < 0.001 for trend; ^(tt)p < 0.05 for trend

TABLE 3 Predicted Positive versus Negative Test Results for Troponin-T.Creatine Kinase-MB, C-reactive Protein and Myeloperoxidase AcuteCoronary MACE 30d MACE 30d Unstable Angina Myocardial InfarctionSyndromes (All patients) (Troponin negative) Variable Ratio χ² Ratio χ²Ratio χ² Ratio χ² Ratio χ² Troponin-T  0:103 38.0** 142:0  604.4**142:103 270.2** 142:103 271.4** 0:0 — Creatine Kinase-MB  5:98 18.0**98:44 273.0** 103:142 120.3** 104:141 121.1**  6:97 0.03 C-ReactiveProtein 24:79 3.5  55:82  5.6*  79:161 0.3  76:164  0.007 21:82 4.3*Myeloperoxidase 67:36 11.5** 96:46  22.2** 163:82   45.0** 161:84  39.7** 65:38 18.1** Additive Value of Markers plus Troponin-T forPrediction of Positive Test Results MACE 30d (All patients) Cut pointsfor variables used were as follows: Troponin-T Variable alone VariablePlus Troponin T (≧0.1 ng/ml). creatine kinase-MB (≧8.8 ng/ml).C-reactive Variable N (%) N (%) protein (≧10 mg/L) and myeloperoxidase(≧198 pM). Troponin-T 142 (58.0) — Note that myeloperoxidase predictsrisk for major adverse cardiac events (MACE) in the ensuing 30 daysCreatine Kinase MB 104 (42.5) 148 (60.4) following presentation to theEmergency Department in troponin negative subjects. Addition ofmyeloperoxidase C-Reactive Protein  76 (31.7) 163 (66.5) to Troponin-Tas a risk stratification strategy also significantly increases thesensitivity for prediction of Myeloperoxidase 161 (65.7) 207 (84.5)major adverse cardiac events. *p < 0.05. **p < 0.001

1. A method of determining if a patient presenting with chest pain is atrisk of experiencing a major adverse cardiac event within the subsequent6 months, comprising: comparing levels of myeloperoxidase (MPO)activity, myeloperoxidase (MPO) mass, or both, in a bodily sample from apatient with chest pain to at least one control value that is based onthe levels of MPO activity, MPO mass, or both in a comparable bodilysample from a control population, wherein the bodily sample is blood,plasma, serum or circulating leukocytes, and wherein a patient whosebodily sample contains elevated levels of MPO activity, MPO mass, orboth as compared to the at least one control value is at risk ofexperiencing a major cardiac event within 6 months of presenting withchest pain.
 2. The method of claim 1, wherein cellular distribution ofmyelopcroxidase levels in the patient's bodily sample is obtained byflow cytometry.
 3. The method of claim 1, wherein said at least onecontrol value is based on the MPO activity levels in comparable bodilysamples from the general population or a select population of humansubjects.
 4. The method of claim 1, wherein said at least one controlvalue is a single representative value or a range of representativevalues and is based on the MPO activity levels in comparable bodilysamples from the general population or a select population of humansubjects.
 5. The method of claim 1, wherein the patient's MPO activitylevel is compared to a plurality of MPO activity level ranges that arebased on the MPO activity levels in comparable bodily samples from thegeneral population or a select population of human subjects.
 6. Themethod of claim 1, wherein the circulating leukocytes are one or more ofthe following: neutrophils, monocytes, sub-populations of neutrophils,and sub-populations of monocytes.
 7. The method of claim 1, wherein thelevels of myeloperoxidase mass in the human patient's bodily sample isobtained by an immunological technique.
 8. The method of claim 1,wherein said control value is based upon the MPO mass levels incomparable bodily samples from the general population or a selectpopulation of human subjects.
 9. The method of claim 1, wherein said atleast one control value is a single representative value or a range ofrepresentative values and is based upon the MPO mass levels incomparable bodily samples from the general population or a selectpopulation of human subjects.
 10. The method of claim 1, wherein thepatient's MPO mass level is compared to a plurality of MPO mass levelranges that are based on the MPO mass levels in comparable bodilysamples from the general population or a select population of humansubjects.
 11. The method of claim 1, wherein a patient whose bodilysample contains elevated levels of MPO activity, MPO mass, or both ascompared to the at least one control value is at risk of experiencing amajor cardiac event within 1 months of presenting with chest pain. 12.The method of claim 1, wherein the sample is blood, serum, plasma, orany combination thereof.
 13. The method of claim 12, further comprisingthe step of determining levels of troponin in the patient's blood,serum, plasma, or any combination thereof.
 14. The method of claim 13,wherein levels of troponin in the patient's blood, serum, and/or plasmaare normal.
 15. The method of claim 13, wherein levels of troponin inthe patient's blood, serum, and/or plasma are elevated.
 16. A method ofdetermining if a patient with chest pain is at risk of experiencing amajor cardiac event within 6 months of presenting with chest pain,comprising: a) obtaining the levels of one or more selectmyeloperoxidase-generated oxidation products in a bodily sample from thehuman patient, wherein said bodily sample is urine, blood, serum, orplasma, wherein each of said select myeloperoxidase-generated oxidationproduct is selected from the group consisting of nitrotyrosine,dityrosine, methionine sulphoxide and an MPO-generated lipidperoxidation product; and b) comparing the levels of each of said selectmyeloperoxidase-generated oxidation product in the bodily sample fromthe human patient with a control value; wherein a patient whose bodilysample contains elevated levels of said one or moremyeloperoxidase-generated oxidation products as compared to the at leastone control value is at risk of experiencing a major cardiac eventwithin 6 months of presenting with chest pain.
 17. The method of claim16, wherein one of said select myeloperoxidase-generated oxidationproduct is nitrotyrosine.
 18. The method of claim 16, wherein one ofsaid select myeloperoxidase-generated oxidation product is an MPO lipidperoxidation product selected from hydroxy-eicosatetraenoic acids(HETEs); hydroxy-octadecadienoic acids (HODEs), F2Isoprostanes; theglutaric and nonanedioic monoesters of 2-lysoPC (G-PC and ND-PC,respectively); the 9-hydroxy-10-dodecenedioic acid and5-hydroxy-8-oxo-6-octenedioic acid esters of 2-lysoPC (HDdiA-PC andHOdiA-PC, respectively); the 9-hydroxy-12-oxo-10-dodecenoic acid and5-hydroxy-8-oxo-6-octenoic acid esters of 2-lysoPC (HODA-PC and HOOA-PC,respectively); the 9-keto-12-oxo-10-dodecenoic acid and5-keto-8-oxo-6-octenoic acid esters of 2-lysoPC (KODA-PC and KOOA-PC,respectively); the 9-keto-10-dodecendioic acid and 5-keto-6-octendioicacid esters of 2-lysoPC (KDdiA-PC and KOdiA-PC, respectively); the5-oxovaleric acid and 9-oxononanoic acid esters of 2-lysoPC (OV-PC andON-PC, respectively); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterolα-epoxide); 5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide);5-cholesten-3β,7β-diol (7-OH-cholesterol); 5-cholesten-3β, 25-diol(25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOHcholesterol); and cholestan-3β, 5α, 6β-triol (triol).
 19. The method ofclaim 16, wherein the level of said myeloperoxidase-generated oxidationproduct in the patient's bodily sample is compared to a representativevalue or a range of representative values that are based upon the levelsof said select myeloperoxidase oxidation product in comparable bodilysamples from the general population or a select population of humansubjects.
 20. The method of claim 16, wherein the control value is aplurality of ranges that are based on the levels of said selectmyeloperoxidase oxidation product in comparable bodily samples from thegeneral population or a select population of human subjects.
 21. Amethod of assessing the risk of requiring medical intervention in apatient who is presenting with chest pain, comprising characterizing thelevels of myeloperoxidase activity, myeloperoxidase mass, or both,respectively in the bodily sample from the human patient, wherein saidbodily sample is blood or a blood derivative, wherein a patient whoselevels of myeloperoxidase activity, mycloperoxidase mass, or both ischaracterized as being elevated in comparison to levels ofmyeloperoxidase activity, myeloperoxidase mass or both in a comparablebodily samples obtained from individuals in a control population is atrisk of requiring medical intervention to prevent the occurrence of anadverse cardiac event within the next six months.
 22. A method ofdetermining whether a patient who presents with chest pain is at risk ofrequiring medical intervention to prevent an adverse cardiac eventwithin the next six months comprising: comparing the level of a riskpredictor in a bodily sample from the subject with a value that is basedon the level of said risk predictor in comparable samples from a controlpopulation, wherein said risk predictor is myeloperoxidase activity,myeloperoxidase mass, a myeloperoxidase-generated oxidation product, orany combination thereof, and wherein said bodily sample is blood, serum,plasma, or urine, wherein a subject whose bodily sample containselevated levels of said risk predictor as compared to the control valueis at risk of requiring medical intervention to prevent an adversecardiac event within 6 months of presenting with chest pain, and whereinthe difference between the level of the risk predictor in the patient'sbodily sample and the level of the risk predictor in a comparable bodilysample from the control population establishes the extent of the risk tothe subject of requiring medical intervention to prevent an adversecardiac event within the next six months.
 23. The method of claim 22,wherein the risk predictor is mycloperoxidase mass or myeloperoxidaseactivity, wherein the sample is blood, serum or plasma, wherein levelsof troponin in the patient's blood, serum, or plasma are normal and,wherein the patient is categorized as being at risk of requiring medicalintervention within the next 6 months based on levels of myeloperoxidaseactivity or myeloperoxidase mass or in the patient's blood, plasma orserum.
 24. The method of claim 22, wherein the difference between thelevel of the risk predictor in the patient's bodily sample and the levelof the risk predictor in a comparable bodily sample from the controlpopulation establishes the extent of the risk to the patient ofrequiring medical intervention within the next 30 days.